DESCRIPTION(Adapted from applicant's abstract): Na+, K+, and Cl- play a key role, both at the level of ligand recognition and substrate translocation by the dopamine transporter (DAT). However, the exact relationships are not known, and there are a number of crucial, unanswered questions that will be addressed in the proposed experiments under the following Specific Aims. First, it will be assessed whether Na+ stimulates DA binding to the DAT through an allosteric effect at a distal site, and what the role is of Cl-. Our previous modeling studies indicated a Na+ site (cation site 2) distal to the cocaine analog binding domain, at which Na+ stimulates cocaine analog binding, and, less certainly, DA binding. Second, substrate-derived inhibitors of the DAT will be studied with differential sensitivity to Na+ and K+ or with preferential affinity for cocaine binding sites vs. DA binding sites. In additions to the distal cation site 2, another site (1) will be studied that overlaps with the DA domain, again including assessment of a role for Cl- for these inhibitors. For a selected number of substrates with differential Na+, K+ sensitivity or with preferential action at DA vs. cocaine binding domains, we will explore the role of sidedness of inhibition at the external andinternal face of the DAT, and assess their cocaine antagonist activity. Third, the order of binding of Na+, Cl- and DA in the transport cycle will be studied in view of the disagreement in the literature as to the sequence in the ordered binding of Na+, Cl- and DA to DAT. Because of the lack of a suitable substitute for Na+, [Na+] will be varied in a novel protocol without a substitute. Classic kinetic [3H]DA uptake experiments and rotating disk electrode voltammetry measurements at varying [Na+] and [Cl-] will be performed and the efflux of preloaded [3H]DA will be examined as a function of Na+ and Cl- in the efflux medium. Fourth, we will search for residues in the DAT involved in Na+ and Cl- sensitivity, by focusing on transmembrane domains (TMs) 1, 3, and 7. Residues in these domains have been implicated in Na+ and Cl- interactions with the transporters for serotonin and GABA which are closely related to the DAT. Specifically, we wish to assess the role of Trp84 and Arg85 in tm1, Glu117 in TM2, Tyr156 and Asn157 in TM3, and Asn353, thr356, Ser357, Ser360, Gly361, Phe362, Phe365, and leu368 in TM7; in addition, Cys90 adjacent to TM1 in the extracellular loop between TM1 and 2 will be studied by site-directed mutagenesis. Combined probing of substrate-derived compounds of structures related to A or residues in the DAT protein may help to define regions in both the substrate structure and the DAT protein that rate to cation interaction. Collectively, the proposed experiments will improve our understanding of ion interactions with the DAT impacting on the functioncof the DAT in translocating substrate.